This grant concerns the mechanism of mRNA degradation by the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus. During lytic HSV infections, the half lives of both host and viral mRNAs are determined largely by the Vhs activity. At early times, Vhs accelerates the degradation of cellular mRNAs, contributing to an overall decrease in host protein synthesis. Following the onset of viral transcription, Vhs accelerates the turnover of viral mRNAs belonging to all kinetic classes. In this role, it helps determine viral mRNA levels and facilitates the sequential expression of different classes of viral genes. Within mRNAs, Vhs appears to initiate degradation near regions of translation initiation. A potential clue to the mechanism of this selectivity comes from the observation that Vhs interacts with the cellular translation initiation factors elF4H and elF4AII. The proposed studies are divided into three specific aims. Aim 1 will characterize the basal Vhs nuclease; that is, the isolated Vhs polypeptide, or a complex of Vhs and elF4H. These studies will define the composition of the basal Vhs endonuclease and compare its cleavage specificity to that which is observed in unfractionated cytoplasmic extracts and infected cells. Aim 2 will involve the genetic characterization of Vhs, focusing upon defining the domains required for interaction with elF4H and elF4AII, and for basal nuclease activity. An emphasis will be to determine whether mutations that abrogate binding to elF4H or elF4AII abolish targeted Vhs cleavage of mRNAs in vivo. Finally, Aim 3 will investigate the mechanism by which the Vhs nuclease is targeted to regions of translation initiation. Experiments will determine whether targeting is dependent upon an mRNA 5' end, ribosome scanning, or the RNA helicase activity of elF4A. Other studies will attempt to reconstitute targeted Vhs cleavage using purified and recombinant factors in vitro. Together these studies should provide significant insights into the mechanism of Vhs-induced mRNA decay.